Various species of mint plants are grown primarily in India, China and Japan commercially for mint oil, menthol, vitamins and other metabolites that are valuable to the pharmaceutical industry. These are used in aromatic oils and herbal medicines. The present invention deals with a tissue culture process for the development of a large number of plants from a specified part of mint plant. The process of the present invention opens up new possibilities for producing somaclonal and physiological variants and for genetic improvement of mints by modern techniques of agrobiotechnology.
Mints are of interest globally because of their valuable secondary metabolites, especially mint oils and menthol for the industry. Since it is a vegetatively propagated crop, mutation techniques have largely been applied to improving characteristics like disease resistance, yield, metabolites and oil content. However, it is possible to apply tissue culture techniques for the improvement of mints by the selection of somaclonal variants and genetic engineering. The main objective of the present invention is to provide a simple process for large scale tissue culture based micropropagation of mints. Another objective of the present invention is to provide a powerful tool for the isolation of physiological variants, somaclones and mutants and for genetic manipulation of mints.
Plant regeneration by tissue culture techniques is well established. A wide varied of plant species has been successfully regenerated in vitro via organogenesis or somatic embryogenesis. Organogenesis leads to organ formation i.e. shoot (or root), which can be isolated to induce development of roots (or shoots) to produce full plants while somatic embryogenesis leads to the development of somatic embryos (embryos developed without genetic fertilization) which have both shoot and root initially and are capable of developing into whole plants. Although the ability of individual parts of plants and cells to regenerate into complete plants (called totipotency) is a well known phenomenon, each plant or plant part requires specialized studies to invent the conditions that allow such regeneration. Some of the factors controlling growth and differentiation of such cultures have been determined. The establishment of interactions among different groups of phytohormones, and growth regulators alone or in combinations are responsible for certain interrelations existing among cells, tissues and organs. So there seems to be a consensus that the success in inducing differentiation depends upon the type of plant part ("explant"), the physiological condition of the explant and physical and chemical milieu of explant during culture. Due to this, the science of tissue culture has been directed to optimize the physiological conditions of source plant, the type of explant, the culture conditions and the phytohormones used to initiate tissue culture. This substantiates the fact that development of a new process for proliferation of plants by tissue culture is not obvious.
One major aspect that has to be investigated on a case-by-case basis is the type of plant growth regulators and the amount of plant growth regulators that induce regeneration. Besides, chemical composition of the medium, temperature and other culture conditions play an important role in the induction of organogensis and somatic embryogenesis and their maturation to healthy fertile plants thereof. The response to medium, hormones and growth conditions differs from plant species to species and variety to variety. Thus inventing conditions for efficient regeneration of plants requires developing specialized knowledge about a given plant.
Another major area where innovativeness is required in tissue culture, is identifying the plant part that efficiently responds to the culture conditions and leads to prolific regeneration. Not all plant parts of a given species are amenable to efficient regeneration. It is a complex combination of the explant selected identified for regeneration, physiological state of the explant, growth conditions and growth regulators that determines success of a plant in tissue culture. Different explants from a given plant usually show entirely different and often unpredictable response to growth conditions for proliferation. No general principles can be applied to achieve regeneration. In each case, identification of the explant and identification of the culture conditions are innovative steps in the development of a tissue culture method for regeneration of a plant part into a number of plants.
To date, regeneration of many species and cultivars of Mentha has been reported through tissue culture. But the processes described earlier are not very efficient. The starting materials (explant) used in the earlier processes were different. For example, these processes utilized axillary buds, leaf pieces and embryos as the starting material. In this respect several reports on tissue culture of mints have been published. Some of these are also related to the establishment of cell suspension cultures and callus, and are listed below for convenience and reference. Application of tissue culture techniques for the production and biosynthesis of useful plant constituents has been exploited for the production of alkaloids from excised root culture, callus and by crown gall tissue in a number of plants. (West F R, Jr and Mike E S 1957. Synthesis of atropine by isolated roots and root callus cultures of ballandona, Botan.Gaz. 119:50-54; Klein R M 1960, Plant tissue culture: a possible source of plant constituents, Econ. Botany 14: 286-289 ). Cell suspension and callus cultures of Mentha piperita & M. spicata were reported to enable the production and biosynthesis of secondary metabolites (Lin and Staba 1961, Peppermint and spearmint tissue cultures, callus formation and submerged culture, Leoydia 24: 139-145; Wang and Staba 1963, Peppermint and spearmint Tissue culture II: Dual-Carboy culture of spearmint Tissue. Jour. of Pharmaceutical Science 52:1058-1062). Such cell suspensions were later reported to biotransform certain precursors into monoterpenes (Aviv D and Gulan E 1978, Biotransformation of monoterpenes by Mentha cell lines: Conversion of pulegone to isomenthone. Planta Medica 33;70-77; Aviv D, Krochmal E, Dantes A and Gulan E. 1983, Biotransformation of monoterpene by Mentha cell lines conversion of pulegone-substituents and related unsaturated .alpha.-.beta. ketones. Planta Medica 47:7-10). Triterpenes were reported to be produced by callus tissue of Mentha arvensis (Karasawa D and Shimzu S 1980, Triterpene acids in callus tissue from Mentha arvensis var. Piperascens.Mol.Agric.Biol Chem.44: 1203-1205). Small quantities of menthol were also detected in suspension cultures and callus cultures with the help of GLC TLC.(Bhaumik C and Dutta P C 1982, Menthol in static and suspension cultures of Mentha arvensis Lin: var piperascens Holmes: Indian Drugs (July) 387-388). These reports did not aim at the regeneration of plants from the above said cultures.
Multiplication of shoots from axillary buds of Mentha spp was reported by tissue culture techniques (Rech E L and Pires M J P 1986. Tissue culture propagation of Mentha spp. by the use of axillary buds. Plant Cell Reports 5:17-18, Revishankar G A and Venkatraman L V 1988. Rapid multiplication of plants from cultured auxillary buds of Mentha piperita. Philippine Jour. of Science 117:121-129). These reports deal with the multiplication of shoots from pre-existing meristems in axis of leaves, and up to 15 shoots could be obtained from single explant of M.viridis, M.pulegium and M.piperita which are reported as highly responding species for tissue culture. In the case of recalcitrant mints, like Mentha arvensis, a few shoots were obtained.
Regeneration of shoots was also reported from leaf explants for M. piperita and M. spicata again, the species known to respond well to tissue culture (Repcakoa K Rychlova G Cellorova B and Honcariv R 1986, Micropropagation of Mentha piperita L. through tissue cultures Herba Hungarica, Tom 25:77-88; Van Eck J M & Kitto S L 1992, Regeneration of peppermint and orangemint from leaf disc. Plant Cell Tiss.Org. Cult. 30:41-49). These leaf based protocols, however are not efficiently reproducible and produce only a few shoots per explant.
Regeneration of shoots from callus cultures of Mentha piperita and M.spicata has also been reported. In these cases, the callus was developed either from mature or immature embryos obtained from developed or developing seeds respectively. Although differentiation of shoots from callus was observed, the efficiency was extremely low and only 5 plants could be regenerated from two out of 65 calli. The explant used in this report i.e. seeds are in paucity because mints are propagated vegetatively (Van Eck J M and Kitto S L 1990, Callus initiation and regeneration in Mentha. Hort. Science 25: 804-806) suggesting that the number of shoots obtained and the success rate is poorer than when explants containing pre-existing meristems were used. Regeneration of M. piperita L. from protoplasts was also reported. The regeneration from protoplasts involves the formation of single isolated cells that multiply to give callus and then differentiate to give shoots. However, the regeneration efficiency was very poor (Sato H, Enomoto S. Oka S, Hosomi K and Ito Y 1993, Plant regeneration from protoplasts of peppermint mentha piperita L. Plant Cell Rep. 12: 546-550; Sato H, Enomoto S. Oka S, Hosomi K, Ito Y, 1994, the effect of 4-1-phenyl N(4 pyridyl) urea on protoplast culture of peppermint Mentha piperita L. Plant Tissue Culture Lett. 11: 134-138). The technique for the regeneration of plants from protoplasts was recently applied to develop interspecific somatic hybrids between peppermint and gingermint by protoplast fusion through electrofusion. The hybrid was confirmed by analysis of oil content, chromosome number and RAPD based DNA analysis (Sato H, Yamada K, Mii M, Hosomo K, Okuyama S, Uzawa M, Ishikawa H, Ito Y, 1996: Production of interspecific somatic hybrid between peppermint and gingermints, Plant Science 115, 101-102).
Wild strains of the bacterium Agrobacterium tumefaciens have been reported to develop crown gall or shooty teratomas on stem of M.piperita. Octopine and succinamopine type T-DNA containing Agrobacterium developed crown galls while nopaline type T-DNA containing Agrobacterium developed shooty teratomas. No attempts were made in these cases to get normal mature plants from such teratomas (Spencer A, Hamill, John D and Rhodes Micheal J C 1990, Production of terpenes by differentiated shoot culture of Mentha citrata transformed with Agrobacterium tumefaciens T37. Plant Cell Rep 8: 601-604).
Table 1 summarizes the state of art of tissue culture processes related to mint plant as covered by patents or described in literature. It is then followed by statement describing the process invented by us in contrast to the known state of art.
TABLE 1 State of art of tissue culture work on Mentha Mode of Report regeneration Phytohormones Explant Remarks 1. Lin & Staba, 1961 Peppermint Callus cultures & BTOA, 2, 4-D, Stem & seeds Culture of stem of and spearmint tissue cultures, submerged culture coconut water peppermint & spearmint callus formation and submerged on medium formed shoot buds and culture, Leoydia 24:139-145 roots at the nodes where preexisting meristems are available. Culture of stem on auxins (BTOA and 2, 4D) containing medium gave callus which could not be regenerated into plants. 2. Wang and Staba 1963, Suspension cultures 2, 4-D Stem Characterisation of Peppermint and Spearmint Tissue spearmint (Mentha Spicata) cell Culture II: dual Carboy culture of inoculated in carboys receiving constant Spearmint Tissue. Journal of air flow and agitation. The effect of Pharmaceutical Science 52:1058- certain antifoam and antibiotic com- 1062 pounds on spearmint tissue growth are discussed. Report does not show regeneration of plants. 3. Aviv D and Galun E. 1978, Suspension culture nil Cells Report indicates that cell Biotransformation of lines derived from monoterpenes by Mentha cell different Mentha lines conversion of pulegone to chemotypes were capable isomenthone planta medica 33: to biotransform pulegone to 70-77 isomenthone. No regeneration of plants. 4. Aviv D, Krochmal E, Dentes Suspension culture nil Cells In this report the A, and Galun E. 1983. biotransformation of Biotransformation of certain compounds with monoterpenes by Mentha cell similarities to pulegone is discussed. lines: conversion of pulegone No regeneration of plants. substituents and related unsaturated .alpha.-.beta. ketones Planta, Medica 47:7-10 5. Karasawa D and Shimizu S. Callus NAA & Kin Stem Describes the effect of 1980, Triterpene Acids in callus NAA and kinetin in media tissues from Mentha arvensis var on the composition of piperascens Mol. Agric. Biol. triterpenes in the callus chem. 44:1203-1205. tissue of Mentha arvensis var piperascens (Japanese mint) & comparison between callus tissue and original plants. Does not show regeneration of plants. 6. Bhaumik C and Dutta P C Callus suspension 2, 4-D, Kin Young leaf Four months old callus 1982, Menthol in static and and isolated cells (fresh) suspension cultures of Mentha including the medium arvensis Lin var pipersens yielded menthol which was Holmes: Indian Drug (July) 387- identified by TLC & GLC 388 techniques. Does not show regeneration of plants. 7. Rech E L and Pires M J P 1986. Regeneration of BAP, Kin Nodal segments A method for rapid Tissue culture propagation of Mentha spp. by multiplication of Mentha Mentha spp. by the use of multiplication of spp. from nodal explant is axillary buds Plant Cell Reports axillary buds discussed. Shoots produced 5:17-18 from preexisting meristems. Process does not work for Mentha arvensis efficiently. 8. Ravishankar G A and Multiplication of 1AA, 2, 4D, NAA, Nodal segments Describes the multiplication Venkataraman L V 1988. Rapid axillary buds & IBA, Kin of shoots via inducing preexisting multiplication of plants from regeneration of meristem in nodal tissue. cultured axillary buds of Mentha plants Only 4 shoots per node were piperita Phillippine Jour of produced by this method Science 117:121-129 for M. pipereta. This report does not work for M. arvensis. 9. Repcakoa K. Rychlova M, Regeneration of BAP, Kin Immature leaf Process of regeneration Cellorova E and Honceriv R Mentha piperita was not good because 1986, Micropropagation of identification of the Mentha piperita L. regenerative explant is not easy. Through tissue cultures. Herba Hungarica, Tom 25:7288 10. Van Eck J M and Kitto S L Regeneration of Coconut water Leaf discs Efficiency of shoot 1992, Regeneration of pippermint Mentha plants via BAP, NAA, TFBA formation is very poor. and orange mint from leaf. Plant organogenesis Only a few shoots can be developed Cell Tissue Organ Culture 30:41-49 from one leaf with lower frequency. 11. Van Eck J M & Kitto S L Callus initiation and NAA, BAP Mature and Regeneration from callus 1990, callus initiation and regeneration of immature embryos raised from mature or regeneration in Mentha. Hort. Mentha plant immature embryo was Science 25:804-806 obtained but differentiation of shoots from callus was very poor. Only 5 plants could be regenerated. Further, the explant i.e. mature or immature embryos is very rare because Mentha is vegetatively propagated crop. 12. Sato H, Enomoto S, Oka S, Isolation & culture BAP, NAA Protoplast isolated Isolated protoplasts were Hosomi K and Ito Y, 1993, Plant of from mesophyll demonstrated to induce cell Regeneration from protoplast of protoplast for cells of Mentha division followed by callus peppermint Mentha piperita L. development of piperita leaves formation & development of Plant Cell Rep. 12:546-550 mature Mentha mature plant. But the plant technique is tricky, tedious & time consuming & cannot be adopted for commercial micropropagation 13. Sato H, Enomoto S. Oka S, Isolation & culture 4-PU, BAP, NAA, Protoplast isolated Isolated protoplasts were Hosomi K, Ito Y. 1994. The of Kin and zeatin from mesophyll demonstrated to give calli & effect of 4-1-phenyl N (4 pyridyl) protoplasts for cells then regeneration of whole urea on protoplasts culture of development of of peppermint plant of Mentha piperita. peppermint Mentha piperita L. mature Mentha leaves But the technique of Plant Tissue Cul. Lett. 11:134- plants protoplast isolation & 138 culture is very tricky, tedious & time consuming and not applicable for commercial micropropagation. 14. Sato H, Yamada K, Mii M, Protoplast fusion & 4PU & BAP and Protoplast of Isolated protoplasts of Hosomi K, Okuyama S, Ozawa regeneration of NAA mesophyll cells peppermint & gingermint M, Ishikawa H, Ito Y. 4, 1996: hybrid plants from were fused by electrofusion Production of interspecific peppermint and method and hybrid was somatic gingermint leaves developed by regenerating hybrid between peppermint and plants from fused protoplast. gingermints. Plant Science 115, The hybrid was confirmed 101-102 by analysis of oil content, chromosome number and RAPD based DNA analysis 15. Spencer A, Hamill, John D Genetic -- Internodal stem Wild strain of the bacterium and Rhodes Michael J (1990, transformation and Agrobacterium tumefaciens Production of terpenes by culture of shoots have been reported to develop differentiated shoot culture of crown gall or shooty teratomas Mentha citrata transformed with on stem of M. piperita. Octopine Agrobacterium tumefacienes and succinamopine type T-DNA T37. Plant Cell Rep. 8:601-604. containing Agrobacterium developed crown galls while nopaline type T-DNA containing Agrobacterium developed shooty teratomas.
Novelties in the Present Invention vis a vis State of Art
The present invention provides for the first time an efficient process for callus mediated organogenesis from an easily obtainable explant of mint plant, giving a large number of mature plants. This is potentially very useful in plant biotechnology for micropropagation, selecting variants and genetic transformation. Further, the invention also provides an improved process for exchange and conservation of disease free mint germplasm. The process of this invention is very simple and is applicable to a wide range of varieties and species of genus Mentha. The process also provides a simple method to alter the composition of essential oil in Mentha plants.
The process of the present invention employs the internodal region (for obtaining fully developed plants) as a starting material (explant), which is different from all the earlier reports (as given in Table 1). The process of the present invention for inducing a high frequency of de novo regenerants leads to whole plant development where the de nova regenerants are from tissues other than preexisting meristems. We could identify an explant that when cultured in suitable medium in the presence of certain combinations of commonly used growth regulators can stimulate a high frequency of differentiation of regenerants. Unlike reports 7 and 8 in Table 1, our process gives a larger number of shoots for all species of Mentha tested. Report 8 in Table 1 gives particularly poor regeneration from Mentha arvensis which is not the case with our process. Unlike reports 9 and 11 in Table 1, the internodal explant used by us is very convenient to obtain.
Earlier art dealing with multiple shoot formation used nodal tissue as the explant which consists of preexisting meristematic tissues in the form of axillary buds. The pre-existing meristematic tissue in such explants, when cultured in the presence of growth regulators starts growing to give a few shoots. The present invention uses internodal explant that does not contain preexisting primordia. The internodal explant gives a large number of shoots when cultured in medium supplemented with sufficient amount of growth regulators. The internodal segment has not been used in any earlier report for the regeneration of plants. Only report 15 given in Table 1 used internodal segment but that was for obtaining teratomas and not normal plants.
The phytohormone combinations and the explants used in the present invention are quite different from those used in any of the reports described in Table 1. The multiple shoot regeneration in our protocol was successful within certain limits of the phytohormone levels. For example, BAP functions efficiently at concentration of 8.88, .mu.M to 88.8 .mu.M with naphthalene acetic acid at 0.54 .mu.M to 5.4 .mu.M. .gamma., .gamma., dimethyl allyl amino purine works at 9.84 .mu.M to 78.4 .mu.M with naphthalene acetic acid 0.54 .mu.M to 5.4 .mu.M, and kinetin works at 9.29 .mu.M to 69.3 .mu.M with naphthalene acetic acid 0.54 .mu.M to 5.4 .mu.M. As described in Table 1 these ranges and combinations of phytohormone have not been used earlier for the development of a process for multiple shoot regeneration in mints.